Cah. Biol. Mar. (2011) 52 : 395-403

Temperature effects on the microscopic haploid stage development of ochroleuca and Sacchoriza polyschides , with contrasting life histories

Tânia R. PEREIRA 1,2,3 , Aschwin H. ENGELEN 1, Gareth A. PEARSON 1, Ester A. SERRÃO 1, Christophe DESTOMBE 2,3 and Myriam VALERO 2,3 (1) CCMAR, CIMAR – Laboratório Associado, Faculdade de Ciências e Tecnologias, Universidade do Algarve, Gambelas, 8005-139 Faro, Portugal. Fax.: +351289800069. (2) UPMC Univ Paris 6, UMR 7144, Equipe Biologie Evolutive et Diversité Marine BEDIM, Station Biologique de Roscoff, F-29682 Roscoff Cedex, France (3) CNRS, UMR 7144, Equipe Biologie Evolutive et Diversité Marine BEDIM, Station Biologique de Roscoff, F-29682 Roscoff Cedex, France. Fax. : +33298292328, [email protected]

Abstract: forests are one of the most diverse and productive ecosystems worldwide. Global climate change and human exploitation threaten the stability of many of these ecosystems. In this study we compare differences in temperature responses during the microscopic haploid stage development between two kelp species in order to test if the annual Saccorhiza polyschides outperforms the perennial Laminaria ochroleuca at the northern limit of range distribution of L. ochroleuca , in Northern Brittany. Germination and mortality, sex ratio, fecundity and reproduction were measured in culture for the two species and under three different temperatures (10, 15 and 25°C). An effect of temperature was found for all traits except the sex ratio. Both species showed no ability to develop gametophytes at the highest temperature of 25ºC and were more tolerant to lower temperatures. S. polyschides showed higher germination rate, higher fecundity and lower mortality than L. ochroleuca during the period of the experiment. In addition, its gametophytes developed earlier than those of L. ochroleuca, a competitive advantage found in all temperature conditions. Germination rate, mortality and fecundity were significantly different between the two species. In addition, the two species showed a structural difference in the development of microscopic stages, with S. polyschides gametophytes occupying a larger area, which is suggested to result in a greater adhesion capacity. In conclusion, the microscopic stage of the annual species S. polyschides had a significant advantage in fitness compared to the perennial L. ochroleuca . This annual opportunistic species may out compete L. ochroleuca , at least in Brittany, the study region, corresponding to its northern limit, in areas where they share habitat.

Résumé : Les forêts de laminaires font partie des écosystèmes les plus diversifiés et les plus productifs au monde. Cependant, leur stabilité est de plus en plus menacée par les changements climatiques et les activités humaines. Le but de cette étude est de comparer la réponse à la température des spores de deux espèces de grandes algues brunes afin de tester si l’espèce annuelle Saccorhiza polyschides dont l’aire de répartition s’étant du Maroc jusqu’à la Norvège, est meilleure compétitrice que l’espèce pérenne Laminaria ochroleuca dans le nord de son aire de distribution, en Bretagne Nord. Les taux de germination, de mortalité, le sexe-ratio, la fertilité et la reproduction de ces deux espèces ont été mesurés en culture dans trois conditions de température (10, 15 et 25°C). Les résultats montrent que la température agit sur toutes ces variables sauf sur le sexe-ratio. Quelle que soit l’espèce étudiée, les gamétophytes supportent les températures basses, mais sont

Reçu le 5 janvier 2011 ; accepté après révision le 19 avril 2011. Received 5 January 2011; accepted in revised form 19 April 2011. 396 TEMPERATuRE EFFECTS ON KELP SPORES incapables de se développer au dessus de 25°C. Les taux de germination, de mortalité et de fécondité sont significativement différents entre les deux espèces. S. polyschides montre pendant la durée l’expérience, des taux de germination et de fertilité supérieurs à ceux de L. ochroleuca mais un taux de mortalité inférieur. De plus, ces deux espèces montrent des différences structurales de développement des gamétophytes avec S. polyschides occupant une plus grande surface, ce qui lui conférerait des meilleures capacités d’adhésion au substrat. En conclusion, le stade microscopique de l’espèce annuelle S. polyschides qui se caractérise par des meilleurs paramètres de croissances et de survie, lui conférerait une meilleure valeur sélective que l’espèce pérenne L. ochroleuca . L’espèce annuelle opportuniste pourrait donc supplanter L. ochroleuca , au moins en Bretagne, dans sa limite Nord de distribution où ces deux espèces sont en sympatrie.

Keywords: Kelp l Temperature l Growth rate l Fecundity l Haploid gametophyte l Demography

Introduction period, originate the macroscopic sporophytes (e.g., Kinlan et al., 2003, Barradas et al., 2011, also reviewed by Carney Changes in climate can strongly alter ecosystems, including & Edwards, 2006). shifting species distribution ranges (Spooner, 1950; Fletcher Kelps have been classified as annual or perennial based & Farrell, 1999; Steneck et al., 2002; Hiscock et al., 2004; only on the macroscopic sporophyte life span. In annual Hampe & Petit, 2005; Müller et al., 2009; Wernberg et al., species, sporophytes are only found during a portion of the 2010). In seaweeds, temperature is recognized as the major year. In contrast, perennial species have macroscopic environmental factor controlling geographic range and depth sporophytes with a life expectancy greater than one year distribution (Breeman, 1988; Izquierdo et al., 2002; Steneck and that reproduces for at least two years (varying between et al., 2002; Müller et al., 2009). 2 and 25 years depending on species; Birkett et al., 1998; Kelp forests are known to be one of the most diverse and Steneck et al., 2002). In both annual and perennial species, productive ecosystems worldwide (Mann, 1973; Steneck et the life cycle is completed by microscopic forms of al., 2002; Wernberg et al., 2010). These large brown unknown longevity. After the decay of annual sporophytes, seaweeds play a fundamental role as structural ecosystem survival of microscopic stages during the sporophyte components, serving as food for herbivores and resting season is critical for the maintenance of the detritivorous animals, and as shelter and nursery for a population, while in perennial species the survival of variety of species (Steneck et al., 2002; Leblanc et al., microscopic stages may become critical only in case of 2011). Their canopy reduces the light, creating favourable massive destruction. Such a case was reported in 1992 and conditions for shade-adapted species, while by reducing 1997/98 in Chile, when the ENSO (El Niño Southern water flow, they also influence sedimentation and erosion Oscillations) caused a rise in seawater temperature, leading rates (Steneck et al., 2002). Finally, kelps are, in general, an to a decrease in reproduction and recruitment of Lessonia economically important human resource, as food, fertilizer nigrescens Bory de Saint-Vincent 1826 (Martínez et al., in agriculture and mariculture, and nutrition for cattle 2003). When such events take place, surviving microscopic (Braud, 1974; Peteiro et al., 2006, Sousa-Pinto & Araújo, stages may allow rapid recolonization of the affected areas 2006). Kelp extracts are used in dye, textiles, cosmetics and (Ladah & Zertuche-Gonzaléz, 2007). pharmaceutics, and as a thickener in food preparation Analogous to recolonization of open gaps in a forest, (Braud, 1974). annual species are expected to be the pioneer colonizers, The kelp life cycle consists of a microscopic haploid playing the role of opportunistic species as shown by gametophyte phase, alternating with macroscopic diploid Peteiro et al. (2006), who highlighted that Saccorhiza sporophytes. These release meiotic spores that settle on the polyschides (Lightfoot) Batters was the fastest-growing substrate. Spores germinate and develop into dioecious alga and was able to colonize newly available space in a male and female haploid gametophytes, producing biennial culture of Saccharina latissima (Linnaeus) C.E. antheridia and oogonia, respectively. After fertilization and Lane, C. Mayes, Druehl and G.W. Saunders. Competitive syngamy, the diploid zygote originates a new sporophyte interactions of this nature raise concerns for the resilience (Dayton, 1985). The gametophyte phase and subsequent of such a complex ecosystem in places where human sporophyte embryos are thought to be part of a “bank of activity and climate change might increase disturbance microscopic forms” that will, in the following favourable levels, causing shifts in species composition. Problems T. R. PEREIRA, A. H. ENGELEN, G. A. PEARSON, E. A. SERRÃO, C. DESTOMBE, M. VALERO 397 include kelp species unable to recolonize areas along L. ochroleuca is a Lusitanian species, found very deep in disturbed coasts (e.g. Coleman et al., 2008); or after Azores (Tittley & Neto, 2000) and the Gorringe submerged repeated harvesting at the same location (Engelen et al., bank, and in the warm and temperate waters from Morocco 2011) and regression of kelp distributional limits (e.g. Assis to the Portuguese and North-West Spanish coasts, as well as et al., 2009; Wernberg et al., 2010). very deep in some areas in the Mediterranean. It can also be This study was developed with two species with found from Brittany (France) to the English and Bristol contrasting sporophyte life spans, but similar geographical channels (Braud, 1974; van den Hoek, 1982; Birkett et al., and depth distributions: Laminaria ochroleuca De La 1998; Bartsch et al., 2008). S. polyschides shares this Pylaie with perennial sporophytes and Saccorhiza distributional range, but also extends further north along polyschides with annual sporophytes. Their upper the South and West coasts of England, Wales, Scotland and temperature limits are very close, 25ºC for L. ochroleuca Ireland, reaching the west coast of Norway (Norton, 1977; and 24ºC for S. polyschides (tom Dieck & de Oliveira, Birkett et al., 1998). The study site is, thus, the northern 1993). Data for minimum survival temperature were not geographical limit of L. ochroleuca and the central found in earlier reports. However, it is reported that S. distributional range of S. polyschides . polyschides and L. ochroleuca are exposed to an average The depth at which kelps are able to live varies widely winter temperature of 4ºC in Norway and 10ºC in the with the local conditions, particularly temperature (kelp are English Channel, respectively (Braud, 1974; Norton, 1977; typically cold-adapted species) and light. Their deeper limit van den Hoek, 1982). Taking this into account, it is is usually determined by light availability. In clear waters, expected that S. polyschides has a more efficient as the whole light spectrum is able to reach deeper areas, development at low temperatures than L. ochroleuca . kelps may grow below 35 m. More turbid waters will This experimental work aimed to predict what would be reduce light penetration and change its spectral the future composition of the kelp forests in the study area composition, preventing the kelp from developing at higher in case of a massive destruction or a rise in water depths (Van Den Hoek, 1982; Birkett et al., 1998) temperature. As such, differences in development and fitness were sought in two species that share this same Culture habitat but have contrasting life histories: L. ochroleuca , a Reproductive tissues of both L. ochroleuca and S. perennial species, and S. polyschides , an annual. To answer polyschides were collected at Morlaix Bay (Brittany, this question, (1) differences in development between the France) in July 2009, from depths around 5 m. On the day two species were investigated, (2) developmental rates of of collection, the 15 most reproductive individuals of each both species at temperatures within the upper range of nat - species were chosen, and divided in three sets with 3 tubes ural exposure were estimated, (3) the responses of each each. This way, all tubes in the same set had spores from the species to the highest temperature at their geographical same 5 individuals. After washing the tissue with filtered southern limit were assessed and, finally, (4) differences in seawater (SW), sporulation was initiated and lasted 15 to fertility were quantified. 18 h during which the spores settled on a microscope glass slide placed inside a 50 mL falcon tube filled with filtered Materials and Methods SW (Oppliger et al., 2011), on a shaker at 100 rpm. The following day was considered the first day of culture. The SW was replaced by 0.2 µM Provasoli Biological material Enriched Seawater (Provasoli, 1968), with medium changes every five days. One tube from each set was placed Growth of L. ochroleuca sporophytes is seasonal and usu - at 10, 15 and 25ºC. A 12:12 h light:dark photoperiod and ally takes place during the winter/spring. In photon fluence rate of 30 µmol m -2 s-1 were used. The summer/autumn, blade growth is reduced and spore experiment lasted 26 days and counts were made on day production is maximum (Lüning et al., 2000). The one, two, and every two days subsequently. Each slide had sporophyte of S. polyschides is annual. The growth of the reference points to ensure that the same area was always juvenile blade starts in the spring and maximum size is studied. reached in the summer. In the early autumn, sporophytes To assess spore development, the following different life reach reproductive maturity. unlike L. ochroleuca , the history phases were distinguished based on their spores are generated at the base of the frond, which breaks morphology (Figs 1 & 2): non germinated settled with the first winter storms. Microscopic gametophyte meiospores (Ng), germinated spores with one cell (identi - stages develop from the spore, overwinter, and generate fied by the presence of a germination tube) (1C), macroscopic sporophytes in the following spring (Mann, germinated spores with two cells (2C), germinated spores 1973; van den Hoek, 1982). with more than two cells (+2C), male gametophytes (M), 398 TEMPERATuRE EFFECTS ON KELP SPORES

general linear model (3-way ANOVA) to test whether they differed significantly between species (fixed factor, 2 levels), temperature (fixed factor, 3 levels) or time (fixed factor, 14 levels: 1, 2, 4, 6, 8, 10... 26). Significance was considered for p-values < 0.05. Pairwise differences for significant interaction terms were analysed using Tukey tests. Death and germination rate can only be considered while Figure 1. Overall scheme of spore developmental phases, there are still live forms and spores available for germination, based on morphology. The non dashed arrows indicate the respectively. Thus, to study these parameters, only the days transition that may occur between observations. Ng: non satisfying these conditions for both species and all the germinated settled meiospores; 1C: germinated spores with only temperatures were considered. All statistical analyses were one cell; 2C germinated spores with two cells; +2C: germinated carried out with Minitab (State College, PA, uSA). spores with more than two cells whose gender was not recognizable yet; M: male gametophytes; Fg: female non mature gametophytes; Fm. Female mature gametophytes; Fs: female Results gametophytes with sporophyte. The dashed arrow indicates the contribution of the male gametes to originate the sporophyte that is adhering to the female at the Fs stage. Germination Figure 1. Schéma des stades de développement des spores, basé sur leur morphologie. Les flèches non pointillés indiquent la Germination rate varied temporally between species and transition qui peut survenir entre stades. Ng : meiospores non among temperatures (3-way ANOVA, Species x Time, p < germées fixés; 1C : spores germées avec une seule cellule ; 2C: 0.001 and Temperature x Time, p < 0.05, Figs 3 & 4, spores germées avec deux cellules ; +2C : spores germées avec respectively and Table 1). At day two, S. polyschides plus de deux cellules ; M : gamétophyte mâle ; Fg : gamétophyte femelle immature ; Fm : gamétophyte female mature ; Fs: gamé - showed a significantly higher germination rate than L. tophyte femelle avec sporophyte. Les flèches en pointillés ochroleuca (Tukey test, p < 0.001) and both species had a indiquent la contribution des gamètes mâles à l'origine des higher germination rate compared to the following days sporophytes qui restent sur la femelle correspondant au stade Fs. (Tukey test, Species x Time, p < 0.001 and p < 0.01, S. polyschides and L.ochroleuca , respectively, Fig. 3). No significant difference was found between days 4 and 6 and immature female gametophytes (Fg), mature female game - between both species (Tukey test, Species x Time, tophytes (Fm) and female gametophytes with sporophyte p=0.665). (Fs). unicellular or multicellular stages were distinguished On day two, germination rate of both species, combined, as the growth may be influenced by environmental factors, was significantly higher at 15ºC (Tukey test, p < 0.001). such as temperature (Izquierdo et al., 2002). Mortality Statistical analysis Mortality differed between species depending on Frequencies of all stages of development were calculated temperature (3-way ANOVA, Species x Temperature, p < relative to the total number of spores at day 1. Fecundity 0.001, Table 1). However, this difference was due to the and reproductiveness were calculated as follows: higher mortality at 25ºC of L. ochroleuca showing 4 times higher death rate than S. polyschides (Fig. 5, Tukey test, Mature females + Females with sporophyte p<0.001). For S. polyschides , mortality increased with Fecundity = increasing temperature with 4 times higher death rate at Total females 25ºC compared to 10ºC (Tukey test, p < 0.05), whereas for Females with sporophyte Reproductiveness = L. ochroleuca mortality was not significantly different Total females between 10 and 15ºC (Tukey test, p = 0.98), but about 7 times higher at 25ºC (Fig. 5, Tukey test, p < 0.001). No Total males Sex ratio = temporal variation was detected (3-way ANOVA, time, p = Total females 0.967). At 25ºC, mortality was complete after spore germination for both species obstructing the formation of Frequencies of all stages, germination, death and sex gametophytes. Consequently measures such as sex ratio, ratio, fecundity and reproductiveness from the two species fecundity and reproductiveness of gametophytes were only and under different temperatures were subjected to a analysed for the two lower temperatures. T. R. PEREIRA, A. H. ENGELEN, G. A. PEARSON, E. A. SERRÃO, C. DESTOMBE, M. VALERO 399

Table 1. 3-way ANOVA and significance values for the effect of species, temperature, time and interactions between these on development traits Tableau 1. ANOVA et valeurs de signification pour les effets espèce, température, temps et les interactions sur les traits de développement..

Variables germination rate mortality male/female fecundity reproductiveness

Species < 0.001 < 0.001 0.403 ns 0.002 0.133 ns Temperature < 0.001 < 0.001 0.107 ns 0.006 0.002 Time < 0.001 0.967 ns < 0.001 < 0.001 < 0.001 Species x Time < 0.001 0.742 ns 0.932 ns < 0.001 0.346 ns Temperature x Time 0.042 0.268 ns 0.153 ns 0.126 ns 0.071 ns Species x Temperature 0.092 ns < 0.001 0.141 ns 0.745 ns 0.459 ns Species x Temperature x Time 0.168 ns 0.650 ns 0.203 ns 0.542 ns 0.998 ns

Figure 2. Distinct developments of L. ochroleuca and S. polyschides : o oogonia; a antheridia; f fecundity; s sporophyte (not at scale). Figure 2. Développements distincts de L. Ochroleuca et S. polyschides : o oogonie ; a : antheridia ; f : fécondité ; s sporophyte (non à l’échelle).

Male and Female gametophytes over time from 0.16 at day 10 to 1.12 (mean of the two species and two temperatures) at day 26, when only game - In both species and for both temperatures, male gameto - tophytes were present (data not shown). phytes appeared at the same time as female, as such, when the female gametophytes became mature, there were Fecundity and reproductiveness already males present. Gametophytes of S. polyschides appeared earlier (day 12 at 10ºC and day 10 at 15ºC) than Fecundity varied between species over time (3-way for L. ochroleuca (day 16 at 10ºC and day 12 at 15ºC, Fig ANOVA, Species x Time, p < 0.001, Table 1 & Fig. 7), with 6). Sex ratio showed no differences between species or higher fecundities for S. polyschides than for L. ochroleuca among temperature (3-way ANOVA, species, p = 0.403 and from day 18 to day 22 (Tukey test, p < 0.05), after which temperature, p = 0.107, table 1), but significant temporal there were no significant differences (Tukey test, p = variation was detected (3-way ANOVA, time, p < 0.001). 0.218). In addition, fecundities were 1.4 times higher at Post-hoc comparisons failed to detected temporal 15ºC than at 10ºC (3-way ANOVA, temperature, p < 0.01). differences (Tukey test, Times per species x Temperature p Reproductiveness was 9 times higher at 15ºC compared = 0.074), but the general trend showed increasing sex ratio to 10ºC (3-way ANOVA, temperature, p < 0.01) and 400 TEMPERATuRE EFFECTS ON KELP SPORES

Figure 3. Germination rate of S. polyschides (Sp) and L. Figure 4. Combined germination rate of S. polyschides and L. ochroleuca (Lo) over time (combined temperatures). Letters were ochroleuca at 10, 15 and 25ºC at days 2, 4 and 6. Different letters attributed based on the results of Tukey test, and refer to refer to significant differences between mean values. Based on significant differences between mean values (p < 0.05) Tukey test (p < 0.05). Figure 3. Taux de germination de S. polyschides (Sp) et L. Figure 4. Taux de germination moyen de S. polyschides et L. ochroleuca (Lo) au cours du temps (ensemble des températures). ochroleuca à différentes températures. Les lettres indiquent les Les lettres indiquent les différences significatives entre moyennes différences significatives entre moyennes selon le test de Tukey selon le test de Tukey (p < 0,05). (p < 0,05).

Gametophyte development showed temporal variation. (3-way ANOVA, time, p < 0.001), with no interaction between the main effects (3- The life cycle differences between L. ochroleuca and S. way ANOVA, Temperature x Time, p = 0.071). No polyschides extend to their gametophyte development. difference between the species was detected (3-way Male gametophytes were multicellular for both species. ANOVA, species, p = 0.133) However, the male S. polyschides had a larger surface area per volume contacting with the substrate when compared

Figure 5. Mortality of L. ochroleuca (Lo) and S. polyschides (Sp) at 10, 15 and 25ºC. Different letters refer to significant Figure 6. Ratio between males and females over time for S. differences between mean values based on Tukey test (p < 0.05). polyschides (Sp) and L. ochroleuca (Lo) at each temperature Figure 5. Mortalité de L. ochroleuca (Lo) et S. polyschides conditions. (Sp) à 10, 15 et 25°C. Les différentes lettres se réfèrent aux Figure 6. Ratio entre les mâles et femelles au cour du temps différences significatives des valeurs moyennes, test de Tukey pour S. polyschides (Sp) et L. ochroleuca (Lo) à chaque condition (p < 0,05). de température. T. R. PEREIRA, A. H. ENGELEN, G. A. PEARSON, E. A. SERRÃO, C. DESTOMBE, M. VALERO 401

possible explanation for this absence is that ecotype differentiation occurs along the distribution range, as has been described for several kelp species (Bolton & Lüning, 1982; Gerard & Du Bois, 1988; Peters & Breeman, 1993). However, to address the question of local adaptation, more than one site would have to be studied for each species, as was done by Bolton & Lüning (1982) for (Gunnerus) Foslie, L. digitata (Hudson) J.V. Lamouroux and Saccharina latissima , and by Gerard & Du Bois (1988) for Saccharina latissima . There are comparable examples of local adaptation of kelp populations. However, in such examples, the ecotypes that were described are now recognized as different species. For example, Martínez (1999) reveals the occurrence of Figure 7. Fecundity of S. polyschides (Sp) and L. ochroleuca different ecotypes characterized by difference in survival of (Lo) (combined temperatures) over time. the microscopic stages to temperature, between the kelp Figure 7. Fécondité de S. polyschides (Sp) et L. ochroleuca Lessonia nigrescens located in the Central and Northern (Lo) (températures combinées) au cours du temps. part of the Chilean Coast. However, these two ecotypes correspond probably to two different sibling species that have been shown recently to have a disjoint geographical with L. ochroleuca . This was mainly due to differences in distribution (Tellier et al., 2009). The same was found for the arrangements of the cells. S. polyschides males have a Saccharina latissima (as Laminaria saccharina ) see Gerard predominantly linear arrangement of cells, whereas L. & Du Bois (1988) ochroleuca males are aggregated in a ball-like shape (Fig. Although these species have roughly similar southern 2). Female S. polyschides gametophytes developed a multi - distribution boundaries, L. ochroleuca was found to be less cellular structure with large cells, in contrast to L. adapted to the higher temperature than S. polyschides , ochroleuca, in which female gametophytes were composed showing a 4 times higher mortality. In the Strait of Messina of a single large cell (Fig. 2). Female gametophytes of both species produced only a single sporophyte. (Mediterranean), where the highest temperature of their geographic distribution area is felt, it is reported that these species have different depth distribution, with L. Discussion ochroleuca found deeper than 30 m and S. polyschides being able to grow almost until the surface, thus being exposed to different temperatures (van den Hoek, 1982). Temperature effect Van den Hoek (1982) proposed two explanations for this L. ochroleuca is known to have a northern distributional difference in depth distribution: one was that L. ochroleuca boundary caused by the 10ºC winter isotherm (van den was forced to occur at higher depths because of occasional Hoek, 1982). It has also been reported that this species has increase of water temperature over 23ºC. Another or a temperature optimum for spore development between 12 additional possibility is that it is a more shade tolerant and 18ºC (Izquierdo et al., 2002). Thus, a better species. The precise distributional patterns of these species development at 15ºC was expected. support the former hypothesis. Along the Portuguese coast, Contrarily, the distribution of S. polyschides is delimited L. ochroleuca is now only found along the northern to in the North by temperatures of 3ºC and its spores seem to central regions, with S. polyschides extending further south develop more efficiently at temperature between 5ºC and (Assis et al., 2009). Indeed, reports of occurrence of L. 17ºC (Norton, 1977). It was thus not expected to find ochroleuca further south are mainly very deep populations, significant variation between 10 and 15ºC. The difference occurring below 40 m depths (e.g., Azores, Gorringe bank). in development we observed for S. polyschides between 10 Species with the same geographical boundaries might be and 15ºC might be due to an adaptation to the local delimited by different factors and/or in different life phases environment. L. ochroleuca spores, as well as those of S. (Breeman, 1988). As such, one of these kelp species could, polyschides , are reported to have a maximum development for example, be delimited by its winter growth temperature of 23-24ºC for the sporophytes and 25ºC for requirements and the other by summer reproduction the gametophytes (tom Dieck & de Oliveira, 1993; Birkett limitations (Breeman, 1988; Birkett et al., 1998). In et al., 1998). Thus, we did not expect a total absence of addition, kelp sporophyte’s heat tolerance varies along the gametophyte development at 25°C for either species. A year (Lüning, 1984). 402 TEMPERATuRE EFFECTS ON KELP SPORES

No significant effect of temperature on sex-ratio was distributional limit and rendering it more vulnerable to detected in this study while temperature-dependent sex local extinction. ratio was reported in several kelp species (in Laminaria religiosa : Funano, 1983; L. variegata : Nelson, 2005 and Acknowledgments Lessonia nigrescens complex: Oppliger et al., 2011). The authors suggested that under favorable conditions, We thank the Station Biologique de Roscoff’s diving team mortality will be at a minimum and sexes will be present in for collecting fresh fertile L. ochroleuca and S. polyschides equal proportion, while under stress conditions the more and L. Valeria Oppliger for sharing her knowledge in kelp vulnerable sex will suffer the higher mortality suggesting a spore’s culture. Tânia R. Pereira and Aschwin H. Engelen difference in sex ratio. More experiments are needed to were supported by fellowships from the Portuguese Science carefully address this question since interestingly the two study species might respond differently to temperature. and Technology Foundation (FCT). The research was Although not significant, a slight tendency showing an supported by the French National Research Agency ANR increased of males in L. ochroleuca and an increase of ECOKELP (ANR 06 BDIV 012), and FCT projects. females in S. polyschides with increasing temperature from 10 to 15°C was reported in our study. References

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